The aim of this research is to image the expression of choline kinase (ChoK) in breast tumors using near-infrared (NIR) fluorescent choline analogs capable of disrupting lipid metabolism in cancer cells. Breast cancer is the most prevalent cancer affecting women, and while many breast cancer patients respond well to the current standard of care, there are still subtypes of the disease resistant to existing therapies. Although early diagnosis improves the prognosis for these patients, there is a shortage of non-invasive methods to identify biomarkers associated with these malignant tumors. ChoK is an oncogene that is overexpressed in 39% of breast cancers, and is a marker of tumor aggressiveness. Increased ChoK expression is correlated with breast cancer tumor grade and the expression is inversely proportional to estrogen receptor status. A number of anticancer drugs specifically targeting ChoK have been produced. One of these inhibitors, TCD-717, is now being tested in Phase I Clinical Trials for treatment of advanced solid tumors. The development of methods to detect the expression of this oncogene in breast cancer would be highly significant for non-invasive staging of breast cancer status and for the identification of patients suited for ChoK-targeted therapy. Near-infrared (NIR) optical imaging is a relatively inexpensive technique that employs sensitive tracers, without the use of ionizing radiation, to report specific molecular interactions in vivo. The most effective known inhibitor of ChoK in animal models, MN58b, bears close structural similarity to the cyanine dyes, the major class of compounds that are used for NIR optical imaging. This project seeks to exploit this similarity to design NIR-fluorescent choline analogs that effectively inhibit ChoK at concentrations comparable to MN58b. Our prototype compound, JAS239, has a number of favorable characteristics: it enters cancer cells independently of the choline transporters, localizes to the cytoplasm where ChoK is normally expressed, accumulates in non-hypoxic regions of animal tumors, and is effectively reversed by salvage with excess choline. The proposed work will include synthetically fine-tuning the structure of the prototype NIR ChoK analog to optimize the inhibitory properties in vitro and bio-distribution in vivo. Using 14C-choline radio-tracing, NMR of live cells and cell extracts, fluorimetry, and confocal microscopy, the fluorescent choline analogs will be characterized for potency, NIR-fluorescence, and binding selectivity. The top candidates will then be assessed in murine breast cancer models with varying ChoK expression. The diagnostic potential will be explored using trace levels of each fluorophore, with the most specific probes expected to accumulate in the most aggressive tumors due to ChoK-binding. Higher-dose regimens will then be formulated, and tumor growth delay and animal survival measurements compared to MN58b.